Unraveling clonal CD8 T cell expansion and identification of essential factors in γ-herpesvirus-induced lymphomagenesis.

Alcelaphine gammaherpesvirus 1 (AlHV-1) asymptomatically persists in its natural host, the wildebeest. However, cross-species transmission to cattle results in the induction of an acute and lethal peripheral T cell lymphoma-like disease (PTCL), named malignant catarrhal fever (MCF). Our previous findings demonstrated an essential role for viral genome maintenance in infected CD8+ T lymphocytes but the exact mechanism(s) leading to lymphoproliferation and MCF remained unknown. To decipher how AlHV-1 dysregulates T lymphocytes, we first examined the global phenotypic changes in circulating CD8+ T cells after experimental infection of calves. T cell receptor repertoire together with transcriptomics and epigenomics analyses demonstrated an oligoclonal expansion of infected CD8+ T cells displaying effector and exhaustion gene signatures, including GZMA, GNLY, PD-1, and TOX2 expression. Then, among viral genes expressed in infected CD8+ T cells, we uncovered A10 that encodes a transmembrane signaling protein displaying multiple tyrosine residues, with predicted ITAM and SH3 motifs. Impaired A10 expression did not affect AlHV-1 replication in vitro but rendered AlHV-1 unable to induce MCF. Furthermore, A10 was phosphorylated in T lymphocytes in vitro and affected T cell signaling. Finally, while AlHV-1 mutants expressing mutated forms of A10 devoid of ITAM or SH3 motifs (or both) were able to induce MCF, a recombinant virus expressing a mutated form of A10 unable to phosphorylate its tyrosine residues resulted in the lack of MCF and protected against a wild-type virus challenge. Thus, we could characterize the nature of this γ-herpesvirus-induced PTCL-like disease and identify an essential mechanism explaining its development.

Endothelial extracellular vesicles promote tumour growth by tumour-associated macrophage reprogramming.

Tumour-derived extracellular vesicles (EVs) participate in tumour progression by deregulating various physiological processes including angiogenesis and inflammation. Here we report that EVs released by endothelial cells in a mammary tumour environment participate in the recruitment of macrophages within the tumour, leading to an immunomodulatory phenotype permissive for tumour growth. Using RNA-Seq approaches, we identified several microRNAs (miRNAs) found in endothelial EVs sharing common targets involved in the regulation of the immune system. To further study the impact of these miRNAs in a mouse tumour model, we focused on three miRNAs that are conserved between humans and mouse, that is, miR-142-5p, miR-183-5p and miR-222-3p. These miRNAs are released from endothelial cells in a tumour microenvironment and are transferred via EVs to macrophages. In mouse mammary tumour models, treatment with EVs enriched in these miRNAs leads to a polarization of macrophages toward an M2-like phenotype, which in turn promotes tumour growth.

Development of a Specific Anti-capsid Antibody- and Magnetic Bead-Based Immunoassay to Detect Human Norovirus Particles in Stool Samples and Spiked Mussels via Flow Cytometry.

Human noroviruses impose a considerable health burden globally. Here, a flow cytometry approach designed for their detection in biological waste and food samples was developed using antibody-coated magnetic beads. Antipeptide antibodies against murine norovirus and various human norovirus genotypes were generated for capture and coated onto magnetic beads. A flow cytometry assay was then implemented to detect bead-bound human norovirus GI.3 in patient stool samples and in norovirus-spiked mussel digestive tissues. The detection limit for stool samples was 105 gc/mL, thus bettering detection limits of commercially available norovirus diagnosis quick kits of 100-fold; the detection limit in spiked mussels however was ten-fold higher than in stool samples. Further assays showed a decrease in fluorescence intensity for heat- or UV-inactivated virus particles. Overall, we demonstrate the application of a flow cytometry approach for direct detection of small non-enveloped virus particles such as noroviruses. An adaptation of the technology to routine diagnostics has the potential to contribute a rapid and sensitive tool to norovirus outbreak investigations. Further improvements to the method, notably decreasing the detection limit of the approach, may allow the analysis of naturally contaminated food and environmental samples.

Extracellular Vesicles Mediate Communication between Endothelial and Vascular Smooth Muscle Cells.

The recruitment of pericytes and vascular smooth muscle cells (SMCs) that enwrap endothelial cells (ECs) is a crucial process for vascular maturation and stabilization. Communication between these two cell types is crucial during vascular development and in maintaining vessel homeostasis. Extracellular vesicles (EVs) have emerged as a new communication tool involving the exchange of microRNAs between cells. In the present study, we searched for microRNAs that could be transferred via EVs from ECs to SMCs and vice versa. Thanks to a microRNA profiling experiment, we found that two microRNAs are more exported in each cell type in coculture experiments: while miR-539 is more secreted by ECs, miR-582 is more present in EVs from SMCs. Functional assays revealed that both microRNAs can modulate both cell-type phenotypes. We further identified miR-539 and miR-582 targets, in agreement with their respective cell functions. The results obtained in vivo in the neovascularization model suggest that miR-539 and miR-582 might cooperate to trigger the process of blood vessel coverage by smooth muscle cells in a mature plexus. Taken together, these results are the first to highlight the role of miR-539 and miR-582 in angiogenesis and communication between ECs and SMCs.

Macrophage-derived exosomes attenuate fibrosis in airway epithelial cells through delivery of antifibrotic miR-142-3p.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease of unknown aetiology and cure. Recent studies have reported a dysregulation of exosomal microRNAs (miRs) in the IPF context. However, the impact of IPF-related exosomal miRs on the progression of pulmonary fibrosis is unknown. METHODS: Two independent cohorts were enrolled at the ambulatory care polyclinic of Liège University. Exosomes from sputum were obtained from 19 patients with IPF and 23 healthy subjects (HSs) (cohort 1), and the ones from plasma derived from 14 patients with IPF and 14 HSs (cohort 2). Exosomal miR expression was performed by quantitative reverse transcription-PCR. The functional role of exosomal miRs was assessed in vitro by transfecting miR mimics in human alveolar epithelial cells and lung fibroblasts. RESULTS: Exosomal miR analysis showed that miR-142-3p was significantly upregulated in sputum and plasma of patients with IPF (8.06-fold, p<0.0001; 1.64 fold, p=0.008, respectively). Correlation analysis revealed a positive association between exosomal miR-142-3p and the percentage of macrophages from sputum of patients with IPF (r=0.576, p=0.012), suggesting macrophage origin of exosomal miR-142-3p upregulation. The overexpression of miR-142-3p in alveolar epithelial cells and lung fibroblasts was able to reduce the expression of transforming growth factor ß receptor 1 (TGFß-R1) and profibrotic genes. Furthermore, exosomes isolated from macrophages present antifibrotic properties due in part to the repression of TGFß-R1 by miR-142-3p transfer in target cells. DISCUSSION: Our results suggest that macrophage-derived exosomes may fight against pulmonary fibrosis progression via the delivery of antifibrotic miR-142-3 p to alveolar epithelial cells and lung fibroblasts.

Sputum exosomes: promising biomarkers for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease of unknown aetiology which leads rapidly to death. As diagnosis of IPF is complex, we aimed to characterise microRNA (miRNA) content of exosomes from sputum of patients with IPF. Using miRNA quantitative PCR array, we found a substantial dysregulation of sputum exosomal miRNA levels between patients with IPF and healthy subjects and identified a unique signature of three miRNAs. Interestingly, we found a negative correlation between miR-142-3p and diffusing capacity of the lungs for carbon monoxide/alveolar volume. This is the first characterisation of miRNA content of sputum-derived exosomes in IPF that identified promising biomarkers for diagnosis and disease severity.

The interaction of uPAR with VEGFR2 promotes VEGF-induced angiogenesis.

In endothelial cells, binding of vascular endothelial growth factor (VEGF) to the receptor VEGFR2 activates multiple signaling pathways that trigger processes such as proliferation, survival, and migration that are necessary for angiogenesis. VEGF-bound VEGFR2 becomes internalized, which is a key step in the proangiogenic signal. We showed that the urokinase plasminogen activator receptor (uPAR) interacted with VEGFR2 and described the mechanism by which this interaction mediated VEGF signaling and promoted angiogenesis. Knockdown of uPAR in human umbilical vein endothelial cells (HUVECs) impaired VEGFR2 signaling, and uPAR deficiency in mice prevented VEGF-induced angiogenesis. Upon exposure of HUVECs to VEGF, uPAR recruited the low-density lipoprotein receptor-related protein 1 (LRP-1) to VEGFR2, which induced VEGFR2 internalization. Thus, the uPAR-VEGFR2 interaction is crucial for VEGF signaling in endothelial cells.

Endothelial exosomes contribute to the antitumor response during breast cancer neoadjuvant chemotherapy via microRNA transfer.

The interaction between tumor cells and their microenvironment is an essential aspect of tumor development. Therefore, understanding how this microenvironment communicates with tumor cells is crucial for the development of new anti-cancer therapies. MicroRNAs (miRNAs) are small non-coding RNAs that inhibit gene expression. They are secreted into the extracellular medium in vesicles called exosomes, which allow communication between cells via the transfer of their cargo. Consequently, we hypothesized that circulating endothelial miRNAs could be transferred to tumor cells and modify their phenotype. Using exogenous miRNA, we demonstrated that endothelial cells can transfer miRNA to tumor cells via exosomes. Using miRNA profiling, we identified miR-503, which exhibited downregulated levels in exosomes released from endothelial cells cultured under tumoral conditions. The modulation of miR-503 in breast cancer cells altered their proliferative and invasive capacities. We then identified two targets of miR-503, CCND2 and CCND3. Moreover, we measured increased plasmatic miR-503 in breast cancer patients after neoadjuvant chemotherapy, which could be partly due to increased miRNA secretion by endothelial cells. Taken together, our data are the first to reveal the involvement of the endothelium in the modulation of tumor development via the secretion of circulating miR-503 in response to chemotherapy treatment.

PAI-1 mediates the antiangiogenic and profibrinolytic effects of 16K prolactin.

The N-terminal fragment of prolactin (16K PRL) inhibits tumor growth by impairing angiogenesis, but the underlying mechanisms are unknown. Here, we found that 16K PRL binds the fibrinolytic inhibitor plasminogen activator inhibitor-1 (PAI-1), which is known to contextually promote tumor angiogenesis and growth. Loss of PAI-1 abrogated the antitumoral and antiangiogenic effects of 16K PRL. PAI-1 bound the ternary complex PAI-1-urokinase-type plasminogen activator (uPA)-uPA receptor (uPAR), thereby exerting antiangiogenic effects. By inhibiting the antifibrinolytic activity of PAI-1, 16K PRL also protected mice against thromboembolism and promoted arterial clot lysis. Thus, by signaling through the PAI-1-uPA-uPAR complex, 16K PRL impairs tumor vascularization and growth and, by inhibiting the antifibrinolytic activity of PAI-1, promotes thrombolysis.

Impaired angiogenesis and tumor development by inhibition of the mitotic kinesin Eg5.

Kinesin motor proteins exert essential cellular functions in all eukaryotes. They control mitosis, migration and intracellular transport through interaction with microtubules. Small molecule inhibitors of the mitotic kinesin KiF11/Eg5 are a promising new class of anti-neoplastic agents currently evaluated in clinical cancer trials for solid tumors and hematological malignancies. Here we report induction of Eg5 and four other mitotic kinesins including KIF20A/Mklp2 upon stimulation of in vivo angiogenesis with vascular endothelial growth factor-A (VEGF-A). Expression analyses indicate up-regulation of several kinesin-encoding genes predominantly in lymphoblasts and endothelial cells. Chemical blockade of Eg5 inhibits endothelial cell proliferation and migration in vitro. Mitosis-independent vascular outgrowth in aortic ring cultures is strongly impaired after Eg5 or Mklp2 protein inhibition. In vivo, interfering with KIF11/Eg5 function causes developmental and vascular defects in zebrafish and chick embryos and potent inhibition of tumor angiogenesis in experimental tumor models. Besides blocking tumor cell proliferation, impairing endothelial function is a novel mechanism of action of kinesin inhibitors.

Dre-miR-2188 targets Nrp2a and mediates proper intersegmental vessel development in zebrafish embryos.

BACKGROUND: MicroRNAs (miRNAs) are a class of small RNAs that are implicated in the control of eukaryotic gene expression by binding to the 3'UTR of target mRNAs. Several algorithms have been developed for miRNA target prediction however, experimental validation is still essential for the correct identification of miRNA targets. We have recently predicted that Neuropilin2a (Nrp2a), a vascular endothelial growth factor receptor which is essential for normal developmental angiogenesis in zebrafish, is a dre-miR-2188 target. METHODOLOGY: Here we show that dre-miR-2188 targets the 3'-untranslated region (3'UTR) of Nrp2a mRNA and is implicated in proper intersegmental vessel development in vivo. Over expression of miR-2188 in zebrafish embryos down regulates Nrp2a expression and results in intersegmental vessel disruption, while its silencing increases Nrp2a expression and intersegmental vessel sprouting. An in vivo GFP sensor assay based on a fusion between the GFP coding region and the Nrp2a 3'UTR confirms that miR-2188 binds to the 3'UTR of Nrp2a and inhibits protein translation. CONCLUSIONS: We demonstrate that miR-2188 targets Nrp2a and affects intersegmental vessel development in zebrafish embryos.

Zebrafish Sox7 and Sox18 function together to control arterial-venous identity.

Sox7 and Sox18 are members of the F-subgroup of Sox transcription factors family and are mostly expressed in endothelial compartments. In humans, dominant mutations in Sox18 are the underlying cause of the severe hypotrichosis-lymphedema-telangiectasia disorder characterized by vascular defects. However little is known about which vasculogenic processes Sox7 and Sox18 regulate in vivo. We cloned the orthologs of Sox7 and Sox18 in zebrafish, analysed their expression pattern and performed functional analyses. Both genes are expressed in the lateral plate mesoderm during somitogenesis. At later stages, Sox18 is expressed in all axial vessels whereas Sox7 expression is mainly restricted to the dorsal aorta. Knockdown of Sox7 or Sox18 alone failed to reveal any phenotype. In contrast, blocking the two genes simultaneously led to embryos displaying dysmorphogenesis of the proximal aorta and arteriovenous shunts, all of which can account for the lack of circulation observed in the trunk and tail. Gene expression analyses performed with general endothelial markers on double morphants revealed that Sox7 and Sox18 are dispensable for the initial specification and positioning of the major trunk vessels. However, morphants display ectopic expression of the venous Flt4 marker in the dorsal aorta and a concomitant reduction of the artery-specific markers EphrinB2a and Gridlock. The striking similarities between the phenotype of Sox7/Sox18 morphants and Gridlock mutants strongly suggest that Sox7 and Sox18 control arterial-venous identity by regulating Gridlock expression.